A Novel Screening System to Characterize and Engineer Quorum Quenching Lactonases

Area of Science:

• Microbiology
• Biochemistry
• Enzymology

Background:

• N-acyl l-homoserine lactones (AHLs) are key bacterial signaling molecules in quorum sensing.
• Lactonases inactivate AHLs, inhibiting bacterial virulence and biofilm formation.
• Targeting specific bacterial species requires lactonases with high substrate specificity, which are limited in natural diversity.

Purpose of the Study:

• To develop a novel, medium-throughput screening system for evaluating lactonase activity.
• To facilitate the identification and engineering of lactonases with altered substrate specificity.
• To enable selective targeting of bacterial communities by engineering quorum quenching enzymes.

Main Methods:

• A chromogenic assay was developed by coupling N-acyl l-homocysteine thiolactone ring opening with 5,5-dithio-bis-(2-nitrobenzoic acid).
• The assay is compatible with cell lysates and applicable to lactonases from diverse protein families.
• Mutant libraries of GcL lactonase were screened, and kinetic characterization was performed.

Main Results:

• The screening system successfully evaluated lactonase activity and identified GcL variants with significantly altered substrate specificity.
• Specific variants showed up to 1900-fold reduced activity against long-chain AHLs and a ~38-fold increased preference for short-chain substrates.
• The results were corroborated by kinetic characterization of thiolactonase and homoserine lactonase activity.

Conclusions:

• A robust and versatile lactonase screening system has been established, improving the evaluation of enzyme activity.
• This system accelerates the discovery and engineering of quorum quenching enzymes with tailored substrate specificities.
• The developed system will aid in developing strategies for selective bacterial community modulation.